Targeting CPS1 attenuates lung cancer metastasis by regulating EMT through an epigenetic mechanism

<b>Background:</b> Metastasis is the primary cause of cancer-related mortality, and targeting the drivers of this process is a promising strategy to improve patient outcomes. Recent studies have highlighted a role of Carbamoyl Phosphate Synthetase 1 (CPS1), the urea cycle's rate-limiting enzyme, in tumor development. However, its involvement in tumor spreading and metastasis remains unclear. <b>Methods:</b> Transwell assay, wound healing assay and a range of lung cancer metastasis animal models were employed to investigate the impact of genetic knockdown and pharmacological inhibition of CPS1 on lung cancer metastasis both <i>in vitro</i> and <i>in vivo</i>. Quantitative proteomic analysis, RNA sequencing, untargeted metabolomics and targeted metabolomics to urea cycle were conducted to elucidate the underlying mechanisms of CPS1 inhibition. <b>Results:</b> CPS1 was overexpressed in a subset of patients with metastatic lung cancer, and this increased expression correlated with decreased patient survival. Genetic knockdown and pharmacological inhibition of CPS1 significantly reduced the tumor burden and metastasis in mice with the spontaneous (<i>Kras</i> ^G12D/+; <i>p53</i> ^-/-) and induced metastatic lung cancer. Mechanistically, CPS1 overexpression in metastatic cancer cells resulted in excessive fumarate production, an intermediate metabolite in the urea cycle. Fumarate accumulation inhibited TET2 activity and altered <i>miR200a</i> gene methylation to drive epithelial-to-mesenchymal transition (EMT), thereby enhancing cell migration and invasion. Notably, CPS1 inhibition reduced fumarate accumulation and enhanced TET2 activity, which epigenetically upregulated PD-L1 expression. This activation contributed to impaired CD8⁺ T cell function and ultimately promoted tumor immune evasion. To overcome immune evasion, we investigated a combination therapy. Combining a CPS1 inhibitor with an anti-PD-1 antibody demonstrated a synergistic and potent effect, significantly inhibiting both lung tumor growth and metastasis. <b>Conclusions:</b> These findings define a crucial role for CPS1 in lung cancer metastasis. Targeting CPS1 may offer a valuable therapeutic intervention strategy against metastatic lung cancer.